Engineered Recombinant Hagfish Intermediate Filament Proteins: Unraveling Domain Roles in Synthetic Fiber Formation...

Area of Science:

• Biomaterials Science
• Protein Engineering
• Biopolymer Science

Background:

• Hagfish intermediate filament (HIF) proteins (α and γ subunits) form strong dry fibers.
• Understanding the structure-function relationship of HIF protein domains is crucial for biomaterial development.

Purpose of the Study:

• To investigate how different domains of HIFα and HIFγ proteins influence fiber formation and mechanical properties.
• To design and analyze recombinant HIF protein constructs with varied domain combinations for synthetic fiber spinning.

Main Methods:

• Recombinant expression and purification of HIFα and HIFγ constructs with varying N-terminus, C-terminus, and central rod domain (CRD) combinations.
• Spinning of these constructs into dry fibers and subsequent mechanical testing (tensile strength, strain, toughness, elastic modulus).
• Fourier transform infrared-attenuated total reflection (FTIR-ATR) analysis to determine secondary structure content (β-sheet, α-helical/random coil).

Main Results:

• HIFα constructs showed highest tensile strength without termini; termini improved strain and toughness.
• HIFγ constructs exhibited enhanced tensile strength and elastic modulus with only the N-terminus.
• Mixing α and γ constructs generally improved mechanical properties compared to full-length proteins.
• FTIR-ATR revealed CRD's role in β-sheet content and termini's role in α-helical/random coil regions of stretched fibers.

Conclusions:

• Specific protein domains significantly impact the mechanical properties and structural organization of HIF-based fibers.
• Tailoring domain composition allows for the design of recombinant intermediate filament proteins with optimized performance for biomaterial applications.
• Findings offer insights into structure-function relationships for engineering advanced biomaterials from recombinant proteins.